Recombinant human erythropoietin with advantageous glycosylation profile

ABSTRACT

A method for the production of a polypeptide, the method comprising culturing, under conditions which allow for the expression of DNA encoding SEQ ID NO:2 in an eukaryotic host cell, wherein the DNA is in vector pPHOEBE-40-7, and optionally isolating the polypeptide from the culture, is described. A polypeptide obtained by such method and a pharmaceutical composition comprising the polypeptide also are described. A composition for diagnosing anemia comprising such polypeptide and a method of treating anemia caused by lack of erythropoietin also are described.

CROSS-REFERENCE TO RELAYED APPLICATIONS

The present invention claims the benefit of PCT/EP98/05399, filed Aug.26, 1998, which claims a priority date from EP 97115081.8, filed Sep. 1,1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to polypeptides having part or all of theprimary structural conformation of erythropoietin and having an improvedin vivo half-life and biological activity due to a modifiedglycosylation profile. The present invention also provides DNA sequencesencoding the amino acid sequence of said polypeptides operatively linkedto regulatory elements which allow for the expression of said DNAsequence in eukaryotic host cells as well as vectors comprising such DNAsequences. The present invention also relates to host cells comprisingthe aforementioned DNA sequences and vectors and their use for theproduction of the aforedescribed polypeptides. Furthermore, the presentinvention relates to pharmaceutical and diagnostic compositionscomprising the aforementioned polypeptides, DNA sequences and vectors.The present invention also relates to the use of the aforedescribedpolypeptides, DNA sequences and vectors for the preparation ofpharmaceutical compositions for treating all kinds of anaemia caused bya lack of erythropoietin.

2. Description of the Related Art

The erythrocyte is by far the most common type of cell in the blood.When mature, it is packed full of hemoglobin and contains practicallynone of the usual cell organelles. In an erythrocyte of an adult mammal,even the nucleus, endoplasmic reticulum, mitochondria, and ribosomes areabsent, having been extruded from the cell in the course of itsdevelopment. The erythrocyte, therefore, cannot grow or divide; the onlypossible way of making more erythrocytes is by means of stem cells.Furthermore, erythrocytes have a limited life span of about 120 days inhumans. Worn-out erythrocytes are phagocytosed and digested bymacrophages in the liver and spleen, which remove more than 10¹¹senescent erythrocytes in every human being per day. A lack of oxygen ora shortage of erythrocytes stimulates cells in the kidney to synthesizeand secrete increased amounts of erythropoietin into the blood-stream.The erythropoietin in turn stimulates the production of moreerythrocytes. Since the change in the rate of release of newerythrocytes into the blood-stream is observed as early as 1 or 2 daysafter an increase in erythropoietin levels in the blood-stream, thehormone must act on cells that are very close precursors of the matureerythrocytes. The cells that respond to erythropoietin can be identifiedby culturing bone marrow cells in a semisolid matrix in the presence oferythropoietin. In a few days colonies of about 60 erythrocytes appear,each founded by a single committed erythrocyte progenitor cell. Thiscell is known as an erythrocyte colony-forming cell, or CFC-E, and givesrise to mature erythrocytes after about six division-cycles or less. TheCFC-Es do not yet contain hemoglobin, and they are derived from anearlier type of progenitor cell whose proliferation does not depend onerythropoietin. CFC-Es themselves depend on erythropoietin for theirsurvival as well as for proliferation: if erythropoietin is removed fromthe cultures, the cells rapidly undergo programmed cell death.Erythropoietin as other colony stimulating factor is a glycoprotein thatacts at low concentration (about 10⁻¹² M) by binding to specificcell-surface receptors. These receptors belong to a large receptorfamily, the so-called “cytokine receptor family”, whose members areusually composed of two or more subunits, one of which is frequentlyshared among several receptor types. Mature human erythropoietin is aglycoprotein with a molecular weight of 34 to 38 kD and consists of 166amino acids (AS) and the glycosyl residue accounts for about 40% of themolecular weight. Since erythropoietin is required for the renewal oferythrocytes, this hormone is essential for the quality of life,especially of patients, which suffer from anaemia and hypoxia, due toreduced numbers of red blood cells which can be caused by, e.g.,dialysis or through reduction of erythroid precursor cells as aconsequence of therapies based on the suppression of cellularproliferation or by inborne or aquired insufficiency of erythropoietinproduction. The identification of the human gene encoding erythropoietinmade it possible to recombinantly express this protein in heterologoushost cells and to provide sufficient amounts of recombinant humanerythropoietin (rhuEpo) for the treatment of the diseases mentioned.However, apart from the primary structure of the protein the structureof the sugar side chains of the molecule is of particular importance forthe interaction of Epo within the organism. For example, desialylatedEpo shows no effect upon application in animals. It nevertheless bindsto the receptor and stimulates precursor cells. The activity decrease ofasialo-Epo in vivo can be explained by the fact that it is removed inthe liver via receptors with a specificity for galactosyl residues whichare susceptible in desialylated Epo.

The wildtype Epo, which has been used therapeutically, has in somepatients the effect of increasing the blood pressure, which isdisadvantageous in therapy. It is to be assumed that Epo also isintegrated in the blood pressure regulation. Therefore, it is desirableto have proteins with the physiological effect of Epo at one's disposalwhich do, however, not show these undesired properties but whichnevertheless stimulate the differentiation and division rate ofprecursor cells to erythrocytes. A further side effect of Epo found insome patients is the stimulation of the megakaryocytes for the formationof thrombocytes. Therefore, there is potential danger of thrombosisduring the therapy with Epo, which then has to be discontinuedimmediately. In this case, a higher specificity of the Epo used would bedesirable.

Thus, the technical problem underlying the present invention is toprovide rhuEpo having an improved biological activity and in-vivohalf-life compared to naturally occurring and rhuEpo so far available.

BRIEF SUMMARY OF THE INVENTION

The solution to the above technical problem is achieved by providing theembodiments characterized in the claims.

Accordingly, the invention relates to a polypeptide having part or allof the primary structural conformation of erythropoietin possessing thebiological property of causing bone marrow cells to increase productionof reticulocytes and red blood cells and to increase haemoglobinsynthesis or iron uptake, said polypeptide being the product ofeukaryotic expression of an exogenous DNA sequence and having thefollowing physiochemical properties:

(i) the amino acid sequence comprises the amino acid sequence given inSEQ ID No. 1 or any fragment or derivative thereof by way of amino aciddeletion, substitution, insertion, addition and/or replacement of theamino acid sequence given in SEQ ID No. 1, wherein at least one of theconsensus N-linked glycosylation sites is modified to other than aconsensus N-linked glycosylation site;

(ii) it is glycosylated;

(iii) greater 5% of the N-glycan structures are sulfated; and

(iv) the ratio Z* of the total N-glycan charge Z to the number ofN-glycosylation sites is greater than 170.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Biological activity of different concentrations of rhuEpo(WT)and rhuEpo(Gln24)

A. The biologic activities of different concentrations of rhuEpo wt andrhuEpo(Gln24) were tested in a human bone marrow red colony assay.GM-CSF and medium samples were coanalyzed as controls. The number of redcolonies was evaluated after two weeks of cultivation for each sample.

B. The biological activities of different concentrations of rhuEpo wtand rhuEpo(Gln24) of the invention were tested in a human bone marrowred colony assay. Medium samples were coanalyzed as controls. The numberof red colonies was evaluated after two weeks of cultivation for eachsample.

DETAILED DESCRIPTION OF THE INVENTION

Human Epo has one O-linked (at Ser126 of SEQ ID NO: 1) and threeN-linked glycosyl residues (at Asn 24, Asn 38 and Asn 83 of SEQ ID NO:1), which are sialylated¹. It has been demonstrated that propersialylation of these sugar residues is important for the in vivohalf-life and, subsequently, for the efficacy of Epo². Furthermore, ithas been demonstrated that different glycosylation sites of rhuEpo areglycosylated differently concerning the complexity of the sugarstructures and sialylation^(7.8). The analysis of genetically engineeredglycomuteins showed, that elimination of glycosylation site Asn24 of therhuEpo molecule (SEQ ID NO: 1) resulting in the glycomutein rhuEpo(Gln24) (SEQ ID NO: 2) is advantageous for both expression and in vivoefficacy⁴. It has now been surprisingly found that rhuEpos that have amodified glycosylation profile in terms of sulfated N-glycan structuresand N-glycan charge number per glycosylation site have an improvedin-vivo half-life and biological activity compared to rhuEpo or anyother rhuEpo glycomutein so far described^(3.4). The present inventionis based on the finding that the glycosylation profile of the muteinrhuEpo (Gln24) (SEQ ID NO: 2) can even be improved by expression of thecorresponding DNA sequence under the control of the regulatory elementsof the Bovine Papillomavirus 1 (BPV-1) vector pHOEBE 40-7 in ChineseHamster Ovary cell (CHO) cells. Although lacking one N-glycosylationsite, the polypeptide (SEQ ID NO: 2) comprises a total charge numberhigher than rhuEpo(wt), and even higher than previously describedrhuEpo(Gln24)^(4,5,6), resulting in a significantly higher Z*; see TableII of Example 9. The polypeptide of the invention has a high amount ofsialylated/sulfated sugar structures per glycosylation site, reducingthe clearance of the molecule by the liver-specificasialogalactosyl-receptors. Furthermore, the same biological effect of acertain dose of rhuEpo(wt) is obtained with a lower dose of thepolypeptide of the invention; see FIG.1B. Additionally, as human urinaryEpo was found to contain significant amounts of sulfatedoligosaccharides⁹ the polypeptide of the invention resembles moreclosely the human urinary Epo than any of the other rhuEpo so faravailable.

The potential exists, in the use of recombinant DNA technology, for thepreparation of various derivatives of the polypeptide of the invention,variously modified by resultant single and/or multiple amino aciddeletion(s), substitution(s), insertion(s), addition(s) and/orreplacement(s), for example by means of side directed mutagenesis of theunderlying DNA. Recombinant DNA technology is well known to thoseskilled in the art. Included is the preparation of derivatives retainingthe primary structural confirmation of erythropoietin possessing thebiological property of causing bone marrow cells to increase productionof erythrocytes and red blood cells and to increase hemoglobin synthesisor iron uptake retaining the essential glycosylation profile, namelythat greater than 5% of the N-glycan structures are sulfated and theratio Z* of the total N-glycan charge Z to the number of N-glycosylationsites is greater than 170.

In a preferred embodiment the invention relates to the afore-mentionedpolypeptide, wherein at least one of the consensus N-glycosylation sitesis deleted and/or is replaced with (a) different amino acid(s).

In another preferred embodiment, at least one of the consensusN-glycosylation sites is added to the polypeptide as described above.

In a particularly preferred embodiment, the glycosylation site at theamino acid position 24 (Asn) of the amino acid sequence given in SEQ IDNO: 1 is deleted, preferably by replacing the amino acid Asn at position24 of the amino acid sequence shown in SEQ ID NO: 1 with the amino acidGln shown for example in the amino acid sequence of SEQ ID NO: 2.

As described above, the polypeptide of the invention has a ratio Z* ofthe total N-glycane charge Z to the number of N-glycosylation sitesgreater than 170. In a preferred embodiment Z* is greater than 180, e.g.183, preferably greater than 190, e.g. 194.

Furthermore, the present invention relates to a DNA sequence encodingthe amino acid sequence of the aforedescribed polypeptides operativelylinked to regulatory elements of Bovine Papillomavirus 1 (BPV-1) whichallow for the expression of said DNA in a eukaryotic host cell.Experiments which had been carried out in the scope of the presentinvention revealed that when the mutein rhuEpo(Gln24) is expressed inCHO cells via a BPV-1 expression system the glycosylation pattern issignificantly different from mutein rhuEpo(Gln24) expressed in CHO cellsvia the pABE40-7 expression vector; see Table II, C3 and C4/C5. Usingthe BPV-1 expression system, rhuEpo(GIn24) comprises higher sialylationand a new quality of chargement through sulfatation; see Table II.Besides the expected recombinant protein, BPV-1 expression vectors areable to express several further functional activities, e.g. transactingfactors and others, that can modulate the cellular activities of thehost system. It is surprising, however, that it obviously can alsomodulate the pattern of posttranslational modifications, e.g. theglycosylation or sulfatation of rhuEpo(Gln24) in CHO cells.

In a preferred embodiment, the aforementioned DNA sequence furthercomprises regulatory elements of the metallothioneine 1 (MT-1) gene.Further examples of possible regulatory elements are viral regulatoryelements such as SV40 promoter and enhancer elements.

The present invention also relates to vectors, preferably expressionvectors, comprising a DNA sequence as described above.

In a preferred embodiment, the expression vector is BPV-1 vectorpPHOEBE40-7 described in the examples hereinafter.

The present invention further relates to host cells comprising a DNAsequence or a vector of the invention. The DNA sequence or vector of theinvention which is present in the host cell may either be integratedinto the genome of the host cell or may be maintained in some formextrachromosomally. The host cell can be any eukaryotic cell, such asCHO, baby hamster kidney cell, C127I and others. Preferred host cellsare CHO cells.

Another subject of the invention is a method for the production of thepolypeptide of the invention having part or all of the primarystructural conformation of erythropoietin possessing the biologicalproperty of causing bone marrow cells to increase production ofreticulocytes and red blood cells and to increase hemoglobin synthesisor iron uptake, said method comprising culturing a host cell of theinvention, and optionally isolating said polypeptide from the culture.

Depending on the specific constructs and conditions used, thepolypeptide may be recovered from the cells, from the culture medium orfrom both. For a person skilled in the art it is well known that it isnot only possible to express a native polypeptide but also to expressthe polypeptide as a fusion protein or to add signal sequences directingthe polypeptide to specific compartments of the host cell, ensuringsecretion of the polypeptide into the culture medium etc. Preferably,the polypeptide of the invention is purified by affinity chromatography,using a monoclonal antibody specific for the huEpo-receptor binding siteon the rhuEpo molecule¹⁰. Such monoclonal antibodies can be obtainedaccording to conventional methods known in the art.

Thus, the present invention also relates to the polypeptide obtainableby the afore-mentioned method. The polypeptide of the invention ischaracterized by its increased half-life and bioactivity in-vivocompared to the same polypeptide having part or all of the primarystructural conformation of erythropoietin possessing the biologicalproperty of bone marrow cells to increase the production ofreticulocytes and red blood cells and to increase hemoglobin synthesisor iron uptake but which does not have the advantageous glycosylationprofile as described above for the polypeptide of the present invention.

Moreover, the present invention relates to a pharmaceutical compositioncomprising at least one of the afore-mentioned polypeptides, DNAsequences and/or vectors of the invention either alone or incombination, and optionally a pharmaceutically acceptable carrier orexcipient. Examples of suitable pharmaceutical carriers are well knownin the art and include phosphate buffered saline solutions, water,emulsions, such as oil/water emulsions, various types of wetting agents,sterile solutions etc. Compositions comprising such carriers can beformulated by conventional methods. The pharmaceutical compositions canbe administered to the subject at a suitable dose. The dosage regimenwill be determined by the attending physician considering the conditionof the patient, the severity of the disease and other clinical factors.Suitable doses range, for example, from 1,000 to 10,000 units,preferably 3,000 to 6,000 units and are more preferably 4,000 units.Progress can be monitored by periodic assessment of hematocrit, numberof reticulocytes, number of red blood cells, and the patient's generalstate of health. Administration of the suitable compositions may beeffected by different ways, e.g. by intravenous, intraperetoneal,subcutaneous, intramuscular, topical or intradermal administration.

The invention also relates to a diagnostic composition comprising atleast one of the afore-mentioned polypeptides, DNA sequences and/orvectors either alone or in combination, and optionally suitable meansfor detection. Said diagnostic composition may be used for methods fordetecting anti-human Epo antibodies or Epo receptors. The polypeptide ofthe invention comprised in a diagnostic composition may be coupled toany reporter system such as peroxydase or 99 Tc.

In a further embodiment the invention relates to the use of at least oneof the afore-mentioned polypeptides, DNA sequences and/or vectors eitheralone or in combination for the preparation of a pharmaceuticalcomposition for treating all kinds of anaemia caused by a lack oferythropoietin to increase the overall number of functional red bloodcells of an organism, e.g., in renal anaemia. For example, duringdialysis, red blood cells can be destroyed so that dialysis patients canbecome anaemic. Since the kidneys of these patients are ofteninsufficient and non-functional, a proper erythropoietin supply is notguaranteed so that dialysis patients need rhuEpo therapy to provide asufficient rhuEpo level, which then is able to continuously replace thedestroyed red blood cells.

In the pharmaceutical compositions and uses of the invention thepolypeptide of the invention may be coupled covalently or non-covalentlyto carriers, e.g., keyhole limpet hemocyanine and/or any effector systemsuch as ricin or 99 Tc.

The examples illustrate the invention.

Example 1 Construction of Expression Vector pHOEBE-40-7 (rhuEpo(Gln24))

For the expression of erythropoietin in CHO cells an expression systemderived from the BPV-1 expression vector pCES was used¹¹. To optimizethe translational start site according to Kozak¹², an ACC Triplett wasinserted directly upstream of the first ATG codon of the Epo codingsequence by site directed mutagenesis. Then rhuEpo(wt)-coding sequencesof vector pCES (BamHI-BgIII fragment) were exchanged by the Eposequences with the optimal transcriptional start site, resulting inexpression vector pHOEBE40-1. Alternatively, the sequence coding formutein rhuEpo(Gln24), also including the Kozak sequence, was cloned intothe expression vector, resulting in pHOEBE40-7. In further experimentsthe pABE40 expression vector system⁴ was used for the expression in CHOcells. The mutein sequences and the Kozak sequence were obtained by sitedirected mutagenesis¹³ and the sequences had been verified by sequenceanalysis. The BPV-1 expression system pHOEBE40-contains DNA fragments ofdifferent origin. The different elements are

a) a SalI-EcoRI fragment of 2317 bp derived from plasmid pJYM¹⁴,containing sequences of the bacterial plasmid pML-1, including theampicillin resistance gene (Amp) and bacterial origin of replication(E.coli ORI);

b) an EcoR I-BamH I fragment of 2801 bp, containing the mousemetallothionein 1 (MT-1) promoter and the 3′non-coding region of themurine MT-1 gene, in the reverse orientation. Due to the cloningstrategy, the two parts of the MT-1 gene, are separated by a shortpBR322-derived sequence of 31 bp (Hind III-EcoR I) of vector pJYM¹⁴;

c) a BamH I-Bgl II fragment of 766 bp, containing the rhuEpo(Gln24)c-DNA (E40-7)⁴, and the upstream optimized translation start site.

d) a Bgl II - BamH I fragment of 242 bp containing the SV 40 latepolyadenylation signal¹⁵;

e) a BamH I-Sal I fragment of 7953 bp containing the total genomicsequence of the bovine papilloma virus BPV-1 (corresponding to the 100%BPV-1 genome BamH I-fragment)¹⁶. This sequence contains a eukaryoticorigin of replication, located in a fragment of about 100 bp (7900 bp-52bp of BPV-1 genome, adjacent to the Hpa I site at position 1).Furthermore, the sequence comprises open reading frames coding forBPV-1-specific replication factors, transcription factors andtransforming factors. It is suggested that these factors are able tomodulate posttranslational modification processes.

Example 2 Transfection of CHO dhfr⁻ Cells and Detection TransientExpression Levels

CHO dhfr⁻ cells were grown in Dulbecco's modified Eagle's medium (DMEM)containing 10% fetal calf serum (FCS). Transfections of CHO dhfr⁻ cellswere carried out using the method according to Graham and Van der Eb¹⁷.Transient expression and secretion was analyzed 24 , 48, and 72 hoursafter transfection of 10 μg/ml of rhuEpo expression vector DNA(pABE-40-7)⁴. In each transient expression experiment, vector p4EGD¹⁸carrying the coding sequence of a truncated human IgG₁ Fc (rhu IgG₁ Fc)under the control of the SV40 promoter^(19,20) was cotransfected, andthe expression rates of both rhuEpo and rhu IgG₁ Fc were determinedusing specific enzyme-linked immunoassays (EISAs)^(21,10), respectively.The relative secretions of the rhuEpo muteins were standardized on thesecretion rates of rhuEpo(wt) and rhu IgG₁ Fc (see Table I).

TABLE I Transient Secretion of CHO-Derived rhuEpo Muteins 3 4 2Secretion Secretion Absolute Secretion (%) (%) Time 1 (ng/ml) Relativeto Relative to (hours p.t.) rhuEpo Mutein rhuEpo Fc Fc rhuEpo(wt) 24rhuEpo(wt) 2.9 4.9 59.2 100.0 rhuEpo(Gln24) 23.0 13.7 167.9 283.6 48rhuEpo(wt) 7.2 14.0 51.4 100.0 rhuEpo(Gln24) 53.0 21.0 252.4 491.0 72rhuEpo(wt) 9.7 23.0 42.2 100.0 rhuEpo(Gln24) 74.0 31.0 238.7 565.6

Transient transfectants were tested for expression of rhuEpo(wt) andrhuEpo(Gln24) relative to IgG₁ Fc fusion protein as a secreted referenceprotein post transfection (p.t.) at 24 h, 48 h and 72 h, respectively.The average values of triplicate determinations (n=3) of the absolutesecretion levels from each transfection set were averaged. Thevariations of the triplicate values were below 5% for eitherrhuEpo/rhuEpo mutein secretion or rhuEpoRFc secretion (column 2). Fromthese data the rhuEpo expression levels relative to IgG₁Fc (Fc, 100%)expression were calculated on a percent basis for each experiment(column 3). From these values, the rhuEpo(Gln24) secretion levels wereestimated relative to rhuEpo(wt) on a percent basis for each experiment(column 4).

Example 3 Detection of Transient rhuEpo(wt), rhuEpo(Gln24) Mutein andrhulgG₁ Fc Expression Levels

Supernatants of transiently or stably transfected cultures were testedfor rhuEpo(wt) or rhuEpo mutein content by a rhuEpo-specificELISA^(21,10). Based on a polyclonal rabbit antiserum, this assay wascarried out as follows: microtitration plates were coated overnight with500 ng per ml of a rabbit anti-rhuEpo immunoglobulin fraction. Then theplates were washed three times with PBS containing 0.01% of Tween20 andair-dried at 37° C. Before use, the plates were saturated with PBScontaining 0.05% (w/v) of bovine serum albumin (BSA). Then the plateswere washed three times with PBS-Tween20 and incubated with thesupematant samples for 30 minutes. After washing for three times withPBS containing 0.05% (w/v) of BSA, bound rhuEpo or rhuEpo muteins weredetected by peroxidase-labelled rabbit anti-rhuEpo immunoglobulinfraction (Ig-POD). After 30 min of incubation, the microtitration plateswere washed and the remaining peroxidase activity, corresponding tocaptured rhuEpo, was developed, using tetramethylbenzidine (TMB) as asubstrate. Then the reaction was stopped by addition of H₂SO₄, and theplates were measured in a Behring ELISA Processor II (Behringwerke AG,Marburg, FRG).

Supernatants of transiently transfected cell cultures were screened forthe secretion of IgG₁Fc, to determine the relative secretion of thedifferent rhuEpo muteins. For this, microtitration plates were coatedwith goat anti-human Fc polyclonal immunoglobulin fraction. Afterwashing and saturation (see above), the plates were incubated with thesupernatants for 1 hour. Then the captured IgG₁Fc molecules weredetected by goat anti-human Fc antibodies, labelled with peroxidase(POD) as described above.

Example 4 Production of Stable rhuEpo(Gln24) Mutein Expressing CellClones

Cell clones secreting rhuEpo(Gln24) mutein were obtained bycotransfection of the vector pSV2 dhfr expressing thedihydrofolatereductase gene, providing resistance againstmethotrexate¹⁸, together with expression vectors pABE40-1, pABE40-7,pHOEBE40-1, or pHOEBE40-7, respectively. After transfection the cultureswere selected in the presence of methotrexate. After a period of two tothree weeks, cell colonies grew out and single cell clones were clonedby use of cloning cylinders or according to the limiting dilutionmethod. The production of rhuEpo(Gln24) was analyzed as described inExample 2.

Example 5 Upscaling of Production Cell Clones

Cell clones suitable for production were further cultivated and finallyexpanded to roller bottle cultures. To produce erythropoietins, cellsfrom the respective seed lots were expanded in roller bottles toconfluence. Then the growth medium was replaced by serum-free DMEM,which was harvested at the end of the production phase.

Example 6 Purification of rhuEpo(Gln24)

Purified rhuEpo and glycomuteins were obtained by affinitychromatography using monoclonal antibody 146/0056¹⁰ This antibody wascovalently coupled to sepharose CL4B (Pharmacia, Uppsala, Sweden)according to Fibi¹⁰. After elution at pH 2.5 into 1 ml of 1 M Tris-HCI,pH 9.5, the samples were dialyzed against PBS pH 7.0. The proteinconcentration was calculated from the O.D. 280 nm, and the purity of thepreparation was controlled visually after separation in a polyacrylamidegel and subsequent silver staining (Phast System, Pharmacia).

Example 7 SDS-Page and Silver Staining

SDS-PAGE and Western blotting procedures were carried out as describedrecently¹⁰, using the Phast System, except that rainbow marker proteinsand a gold-labelled-antibody procedure (both from Amersham-Buchler,Braunschweig, FRG) were used.

Example 8 Human Erythroid Precursor Colony Assay

Suspensions of human bone marrow cells were prepared from bone marrowspecimens in phosphate buffered saline. Gradient-purified interphasecells of the suspensions were harvested after centrifugation at 2200rotations per minute for 25 min at 12° C. The cells were washed threetimes, resuspended in MEM-alpha medium with supplements and certomycinas an antibiotic. The cell number was adjusted to 1.1×10⁶/ml, and thecells were mixed with 4 ml rhuEpo Medium (19,1 ml FCS/15,2 mlTransferrin/BSA/FeCI₃/13,7 ml MEM-alpha medium), and 0,7 ml Agar (about70° C.). 200 μl of the agar cell suspension were added per well to 24well tissue culture plates, containing dilutions of the test samples.The cultures were mixed with the test samples and incubated for 14 daysat 37° C in an atmosphere containing 7% CO₂ and 10% O₂.

Example 9 Glycoanalysis

The liberation by PNGase F of the N-glycans of rhuEpo was performed asdescribed by Nimtz.⁷. The liberated N-glycan pools were measured byhigh-pH anion-exchange chromatography with pulsed amperometric detection(HPAE-PAD), using the set-up and the optimized standard gradient “S” forsialylated glycans previously described²². The hypothetical N-glycancharge Z was determined as described by Hermentin^(23,24). In brief, thehypothetical N-glycan charge of the rhuEpo samples was gained by

i) liberating the N-glycan-pool of the glycoprotein via PNGase F

ii) measuring the N-glycan pool via HPAE-PAD

iii) calculating the percentage of the areas (A) of the groups of peaks,separated by charge,

iv) multiplying the area% of the peak-groups in the neutral (asialo-,as), monosialo-(MS), disialo- (DiS), trisialo- (TriS), tetrasialo-(TetraS) and pentasialo (PentaS) region by zero (asialo), 1 (MS), 2(DiS), 3 (TriS), 4 (TetraS), and 5 (Sulfated), respectively, and

v) summarizing the respective products.

Thus, Z was defined as the sum of the products of the respective areas(A) in the asialo (as), monosialo (MS), disialo (DiS), trisialo (TriS),tetrasialo (TetraS) and sulfated region, each calculated as thepercentage of the total peak area set equal to 100%, and each multipliedby the corresponding charge:

Z=A _((as))*0+A _((MS))*1+A _((DiS))*2+A _((TriS))*3+A _((TetraS))*4+A_((Penta S or Sulfated))*5

vi) dividing Z through the number of glycosylation sites to create Z*.Z* gives an estimate of the number of charges per N-glycosylation siteand, thus, of the grade of chargement of a glycosylation site.

The results of the glycoanalysis of rhuEpo so far commercially availableand the rhuEpo of the invention (column C4 and C5) are compared in TableII.

TABLE II Comparison of the glycoylation profiles of differentrecombinant human erythropoietins Behring-Mutein Boehringer rhuEpo(Gln24) rhuEpo (CHO) (CHO) pABE-40-7 Organon Amgen* pABE-40 like C3rhuEpo (CHO) rhuEpo (CHO) peak peak pABE-40-like pABE-40-like groupgroup area of peak charge peak charge area (%) charge area (%) chargeintergration group number group number O950579 number O950576 number(peak group) area (%) share area (%) share K.D07 share K.D04 shareasialo 3.1 0 monosialo 6.1 6.0 disialo 15 30.0 6.4 12.8 5.4 10.7 11.323.0 trisialo 32 96.0 20.7 62.1 25.5 76.5 23.7 71.0 tetrasialo 40 160.072.9 291.6 69.2 276.6 55.9 223.0 sulfated n.d. n.d. 0.0 0 N-glycancharge (a) 286 (b) 367 364 323 number Z (total) Z* 95.3 122.3 121.3162.5 Behring-Mutein rhuEpo (Gln24) (CHO) pHOEBE-40-7 Merckle C4 C5rhuEpo (BHK) peak peak pABE-40-like group group Nimtz⁷ area of area (%)charge area (%) charge peak charge peak charge intergration O950576number o950546 number group number group number (peak group) K.D04 shareD.K20 share area (%) share area (%) share asialo monosialo 2.8 2.8 1.81.8 4.7 4.7 n.d. disialo 3.2 6.3 8.6 17.3 14.5 29.0 21.1 42.2 trisialo13.8 41.4 20.2 60.6 33.9 101.7 35.0 105.0 tetrasialo 64.0 256.0 60.6242.4 46.8 187.2 40.9 163.6 sulfated 16.2 81.2 8.7 43.5 N-glycan charge388 366 (c) 323 (d) 311 number Z (total) Z* 194 183 107 103 (a)calculated from Hokke²⁵ (b) calculated from Watson²⁶ (c) average of 4different batches; 1 HPAE-PAD run, each (d) calculated from Nimtz⁷ Z*Total N-glycan charge number Z/number of N-glycosylation sites. n.d. notdetermined

The carbohydrate analysis of rhuEpo, originally described by Sasaki etal.¹ and Takeuchi²⁷ for rhuEpo (CHO) and by Tsuda²⁸ for rhuEpo (BHK),has recently been extended by studies of Hokke²⁵, Watson²⁶ (forCHO-rhuEpo), and Nimtz⁷ (for BHK-rhuEpo). According to a new method, thehypothetical N-glycan charge Z can be determined as described byHermentin^(23,24). It has been demonstrated that this parameter gives anexcellent estimate of the amount of undersialylated N-glycans toproperly sialylated N-glycans. As the glycans of rhuEpo are known toconsist of mainly tetraantennary structures with 0-3 LacNAc repeats, Zshould amount to a hypothetical N-glycan charge number between 300 and400. Indeed, the N-glycan charge of CHO-rhuEpo (Boehringer Mannheim) wasdetermined to Z=364 +/−2 (CV=0.6%) (n=6; three different experimentswith 2 HPAE-PAD runs, each), and the N-glycan charge of BHK-rhuEpo(Merckle) was determined to Z=323 +/−2 (CV=0.7%) (n=4; four differentlots; 4 different experiments; 1 HPAE-PAD run, each); see Table II²⁴.Thus, the smaller Z value of the BHK-rhuEpo from Merckle clearlyreflected the greater share of undersialylated N-glycans: 34% of theN-glycans were missing one and 12% of the N-glycans were missing twoterminal sialic acid residues; the structures consisted of 40.9 %tetrasialylated, 35.0% trisialylated and 21.1% disialylated structures(Nimtz.⁷). These data from the literature allowed to calculate theN-glycan charge to Z=311, which is in good agreement (deviation<4%) withthe N-glycan charge determined according to Eq. 1, supra, i.e., Z=323,using the same rhuEpo (BHK) from Merckle (see Table II). In theCHO-rhuEpo from Amgen the major (>95%) di-, tri- and tetra-antennarystructures were fully sialylated²⁶. Their separation according to chargeof the PNGase F-released N-glycans (using a Glycopak DEAE column)allowed to calculate the N-glycan charge to Z=367, which is in excellentagreement with the glycan charge of the rhuEpo (CHO) from BoehringerMannheim, used in this study (Z=364, see Table II). In contrast, thestudy of Hokke et al.²⁵, investigating CHO-rhuEpo from Organon Teknika,showed that 18-20% of the N-glycans were missing one, and 3% of theN-glycans were missing two sialic acid residues. Their structuralanalysis enabled to calculate the N-glycan charge to Z=286, which issignificantly smaller than the CHO-rhuEpo from Amgen (Z=367) orBoehringer (Z=364); see Table II)²⁴.

References

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5. U.S. Pat No. 5,457,089

6. EP0,409,113B1

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23. PCT/EP96/02319

24. Hermentin, Glycobiology 6 (1996)

25. Hokke, Eur. J. Biochem. 228 (1995), 981

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2 1 166 PRT Homo sapiens 1 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg ValLeu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Ile ThrThr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu Asn Ile Thr Val ProAsp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg Met Glu Val Gly GlnGln Ala Val Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala ValLeu Arg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser Ser Gln Pro Trp GluPro Leu Gln Leu His Val Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser LeuThr Thr Leu Leu Arg Ala Leu 100 105 110 Arg Ala Gln Lys Glu Ala Ile SerPro Pro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr AlaAsp Thr Phe Arg Lys Leu Asp Arg Val 130 135 140 Tyr Ile His Pro Phe ArgGly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr GlyAsp Arg 165 2 166 PRT Homo sapiens 2 Ala Pro Pro Arg Leu Ile Cys Asp SerArg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu GlnIle Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu Asn Ile ThrVal Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg Met Glu ValGly Gln Gln Ala Val Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu Leu Ser GluAla Val Leu Arg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser Ser Gln ProTrp Glu Pro Leu Gln Leu His Val Asp 85 90 95 Lys Ala Val Ser Gly Leu ArgSer Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110 Arg Ala Gln Lys Glu AlaIle Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu Arg Thr IleThr Ala Asp Thr Phe Arg Lys Leu Asp Arg Val 130 135 140 Tyr Ile His ProPhe Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys ArgThr Gly Asp Arg 165

What is claimed is:
 1. A method for the production of a polypeptide,said method comprising culturing, under conditions which allows for theexpression of a DNA encoding amino acid sequence SEQ ID NO:2 in aneukaryotic host cell, wherein said DNA is in a vector pPHOEBE-40-7, andoptionally isolating said polypeptide from the culture.
 2. The method ofclaim 1, wherein said eukaryotic host cell is a Chinese Hamster Ovarycell.
 3. A polypeptide obtained by the method of claim
 1. 4. Anerythropoietin polypeptide produced by expressing DNA encoding aminoacid sequence SEQ ID NO:2 in a host cell, wherein said DNA is in thevector pPHOEBE-40-7 and said host cell is a Chinese Hamster Ovary cell,and wherein the erythropoietin polypeptide consists of the amino acidsequence of SEQ ID NO:2.
 5. A pharmaceutical composition comprising thepolypeptide of claim 3 and a pharmaceutical acceptable carrier.
 6. Acomposition for diagnosing anemia, said composition comprises thepolypeptide of claim
 3. 7. A method for treating anemia caused by a lackof erythropoietin, comprising administering the polypeptide of claim 3to a subject suffering from the anemia in need thereof and reducing theanemia caused by a lack of erythropoietin.